Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes

Supramolecular synthetic transporters are crucial to understand and activate the passage across lipid membranes of hydrophilic effector molecules. Herein, we introduce photoswitchable calixarenes for the light-controlled transport activation of cationic peptide cargos across model lipid bilayers and inside living cells. Our approach was based on rationally designed p-sulfonatocalix[4]arene receptors equipped with a hydrophobic azobenzene arm, which recognize cationic peptide sequences at the nM range. Activation of membrane peptide transport is confirmed, in synthetic vesicles and living cells, for calixarene activators featuring the azobenzene arm in the E configuration. Therefore, this method allows the modulation of the transmembrane transport of peptide cargos upon Z–E photoisomerization of functionalized calixarenes using 500 nm visible light. These results showcase the potential of photoswitchable counterion activators for the light-triggered delivery of hydrophilic biomolecules and pave the way for potential applications in remotely controlled membrane transport and photopharmacology applications of hydrophilic functional biomolecules.


Materials and General Methods
Commercially available reagents were used as received without further purification. All other compounds were synthesized and characterized as described below. The NMR experiments were run on a Bruker Avance III operating at 400 MHz ( 1 H) or 100 MHz ( 13 C). UV-Vis absorption spectra were recorded using a Varian Cary 100 Bio or a Varian Cary 5000 spectrophotometer in quartz or disposable plastic cuvettes with 10 mm optical path. Fluorescence spectra were recorded on a SPEX Fluorolog-3 Model FL3-22 spectrofluorimeter. All aqueous solutions were prepared in 5 mM phosphate buffer pH 7.4 as measured with a Crison basic 20+ pH meter.
The E isomers of both 1 and 2 were obtained by heating the samples at 60 ˚C overnight, while the Z isomers were obtained by irradiation at 366 nm until full conversions as confirmed by UV-Visible spectroscopy after the calculation of the absorption coefficients of both species. Both E and Z isomer solutions were protected from outside sources of light to avoid unwanted isomerization.
Continuous irradiations experiments were conducted in a Spex Fluorolog-2 Model F111 spectrofluorometer equipped with a 150 W Xe lamp or in a custom photochemical reactor equipped with a 200 W Hg-Xe lamp and using bandpass or cut-off filters to isolate the desired wavelengths. The light flux (I0) was determined using as actinometers, ferrioxalate in water for λirr = 365 nm (I0 = 3.2 × 10 -9 mol/ s) and the diarylethene derivative 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene in hexane for λirr = 500 nm (I0 = 9.1 × 10 -9 mol/s). 1,2 The activation energy for the thermal Z-E isomerization obtained by following the interconversion by UV-Vis absorption spectroscopy at three different temperatures. The first-order rate constants, k (s -1 ) for the interconversion at the three different temperatures were calculated and fitted to the Arrhenius equation for the obtention of the activation energy for the thermal conversion.
Confocal fluorescence microscopy images were taken with a Dragonfly spinning disc confocal microscope mounted on a Nikon Eclipse Ti-E and equipped with an Andor Zyla 4.2 PLUS sCMOS digital camera and processed using ImageJ software (v1.52b). Flow cytometry analyses were conducted on a Guava easyCyte BG HT flow cytometer using InCyte (v3.2, GuavaSoft, Millipore). For the MTT viability assay a Tecan Infinite F200Pro plate reader was used and the curve fitting was performed with GraphPad Prism 6 software (v6.01).

2.3.Synthesis of Counterion Activator 2
1.48 g (2 mmol) of 1.1, 0.563 g (1.5 mmol) of 2.2 and 0.25 g (6 mmol) of LiOH.H2O were dissolved in 10 ml of DMSO and were heated to 75ºC under constant stirring for 48h. The reaction mixture was then precipitated two times with diethyl ether and once with ethyl acetate. The final precipitate was filtrated over vacuum, yielding a yellow powder. This was then dissolved in a small quantity of water and further purified by RP-18 chromatography, with the product being eluted with 10% acetonitrile and 90% water.
After evaporation of the solvent, 277 mg of final product was obtained, corresponding to a yield of 18%.

2.4.Peptide Synthesis
Peptide synthesis was performed following the manual Fmoc solid-phase peptide synthesis (SPPS) using Rink Amide resin (loading 0.45 mmol/g). 6 Firstly, the resin was placed in a peptide synthesis vessel and Finally, the collected fractions were lyophilised and stored at -20 °C. Purity and identity were confirmed by analytical HPLC and mass spectrometry.

Synthesis of peptide R4
Peptide R4 ( Figure S11) was prepared following the previous general procedure for peptide synthesis with an overall yield of 27%.

Synthesis of peptide R6
Peptide R6 ( Figure S13) was prepared following the previous general procedure for peptide synthesis with an overall yield of 55%.

Binding Studies
The binding affinities of the counterion activators and peptide complexes were analyzed by indicator displacement assays. The fluorescent dye chosen for these experiments was lucigenin (LCG) due to its complex with the receptor SC4 being well characterized and due to the high variation of emission between its bound and free form. Firstly, the affinity for LCG was calculated for each of the activators, by titrations followed by fluorescence spectroscopy leading to almost full quenching of the emission. For the competitive assays, a concentration of LCG and activator was chosen in order to have approximately 90% of complexed dye and that mixture was then titrated with each of the peptides, leading to the regaining of emission when LCG was release from the complex.        All assays were done up to 48h after the preparation of the vesicles to ensure their integrity and majority of CF still encapsulated. For the dye efflux assays, CF emission was followed throughout the assay, with consecutive additions of the elements of the solution -the initial solution presented only the vesicles at a fixed concentration. A counterion activator aliquot is then added according to the concentration to be tested and the emission is measured to confirm that CF remains encapsulated; some counterion activators can act as detergents at sufficient concentrations. Finally, the peptide in question is added and fluorescence spectra are measured ⊃ until the emission stabilizes and a maximum of release is reached. If the counterion activator has the capacity to activate the transport, CF should be co-transported to the exterior of the liposome with the influx of the counterion-peptide complex. Finally, Triton X-100 is added to the solution to dissolve the vesicles and obtain the maximum amount of CF that could be released from the liposomes and emit in the outside medium.

5.2.Dye Efflux Assays
EC50 and Ymax values were calculated from the values of corrected emission at the plateau that is reached, according to previously reported assays. 7 Light-activated release assays. The light-modulation offered by this novel counterion activator was tested by performing dye efflux assays as previously described, with the addition of an interval in the measurement where the solution was irradiated at 500 nm. The decrease in ζpotential indicates that upon addition of 1 and 2 to a LUV mixture, these incorporate into the liposome membrane. Figure S35 -Example of release assay kinetics with addition of each element marked in the graph. The EYPC-LUV Ɔ CF concentration was maintained at 15 µM throughout these assays, with 50 mM concentration of CF inside the liposomes. The addition of the peptide initiates the transport activation and in the end of the assay, the measurement is normalized by the addition of Triton X-100, which destroys the vesicles and leads to the maximum of CF emission. Figure S36 -Dose-response curves of the activation of release of Carboxyfluorescein by the two calix-azo activators, A) 1 and B) 2, in the presence of the peptide R4 (5 µM). The EYPC-LUV Ɔ CF concentration was maintained at 15 µM throughout these assays, with 50 mM concentration of CF inside the liposomes. The efficient concentration at 50% release (EC50) and maximum release of each of the activators' isomers (E and Z represented by black squares and red circles, respectively) is presented, as well as the respective fitted curves for the trans isomer; no considerable activation was observed for the cis isomers of both 1 and 2. Figure S37 -Dose-response curves of the activation of release of Carboxyfluorescein by the two calix-azo activators, A) 1 and B) 2, in the presence of the peptide R8 (4 µM). The EYPC-LUV Ɔ CF concentration was maintained at 15 µM throughout these assays, with 50 mM concentration of CF inside the liposomes. The efficient concentration at 50% release (EC50) and maximum release of each of the activators' isomers (E and Z represented by black squares and red circles, respectively) is presented, as well as the respective fitted curves.